The group A streptococcus (Streptococcus pyogenes, GAS) is a bacterial pathogen of enormous medical importance to humans, causing a variety of disease syndromes that range in severity from minor to life-threatening. Mga is a DNA-binding protein of GAS that activates the transcription of several key virulence genes in response to changing environmental conditions, likely through interactions with other regulatory components in the cell. The Mga regulon encodes products essential for the survival of GAS in the host, including the antiphagocytic M protein, M-like immunoglobulin binding proteins, the secreted inhibitor of complement, a collagen-like protein, and a C5a peptidase. Thus, Mga provides an excellent model system to study global regulatory networks involved in GAS pathogenesis and how they may interact. However, we currently know very little about Mga, including what domains of the protein are critical for its function and how environmental signals control the Mga regulon. The specific aims of this project are: (1) To identify domains of Mga involved in DNA-binding and characterize their role in targeting specific promoters; (2) To determine a consensus Mga binding element for each of the known promoter sites through identification of specific Mga/nucleotide interactions; (3) To investigate whether domains of Mga interact directly with other bacterial components to transduce environmental signals (i.e., is Mga a two-component response regulator?); (4) To identify additional factors required for the environmental regulation of the Mga regulon and assess their role in global virulence regulation. An attractive feature of this proposal is our ability to study Mga both as a purified protein in vitro and as a native molecule in its GAS background. As such, we will be able to thoroughly address key questions of GAS pathogenesis associated closely with the environmental regulation of the Mga regulon. The overall objectives of this proposal are (A) to undertake a structure/function analysis of Mga and determine the mechanisms by which this key GAS virulence regulator contributes to streptococcal disease, and (B) improve our general understanding of regulatory pathways that broadly control virulence in these gram-positive pathogens.